Jean M Wilson

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• Momtaz, S., Padilla-Rodriguez, M., Flores, P. C., Rulli, N., Lee, N. Y., & Wilson, J. M. (2025). Rab14 promotes Parkin-mediated mitophagy. Molecular Biology of the Cell, 36(Issue 9). doi:10.1091/mbc.e25-06-0271
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  Mitochondrial degradation by mitophagy is essential to maintain cell metabolism; dysregulation can result in the accumulation of damaged mitochondria. Although the Rab family of small GTPase proteins are involved with vesicular trafficking in the endocytic and biosynthetic pathways, Rab-GTPases also have a role in mitochondrial integrity. However, a role for Rab14, a trans-Golgi network (TGN)-endosomal RabGTPase in mitophagy has not been described. In cells knocked down for Rab14, mitochondria acquire an elongated morphology and increased levels of mitochondrial proteins, whereas overexpression of Rab14 decreased these proteins. Furthermore, mito-Keima assays show increased mitophagy upon Rab14 overexpression. Rab14-induced mitophagy is dependent on Parkin expression, as well as TBK1 and PI3K activity, placing it in the Parkindependent mitophagy pathway. 3D-reconstruction shows contact site formation between Rab14 and mitochondria, and inhibition of the TGN kinase PI(4)KIIIβ decreases Rab14mitochondria contact sites and prevents Rab14-mediated mitophagy, suggesting that TGNderived Rab14 vesicles mediate mitophagy. These results suggest that Rab14 promotes mitophagy and plays an essential role in modulating cellular metabolism.
• Cox, C., Wu, M., Padilla-Rodriguez, M., Blum, I., Momtaz, S., Mitchell, S., & Wilson, J. (2024). Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4. PLoS ONE, 19(5). doi:10.1371/journal.pone.0296003
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  Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors β-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and β-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and β-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and β-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:β-catenin.
• Behling-hess, C., Blum, I. R., Cox, C. M., Momtaz, S., Padilla-rodriguez, M., & Wilson, J. M. (2021). Rab22a regulates the establishment of epithelial polarity.. Small GTPases, 12(4), 282-293. doi:10.1080/21541248.2020.1754104
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  Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.
• Momtaz, S., Molina, B., Mlera, L., Goodrum, F., & Wilson, J. M. (2021). Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection. Journal of virology.
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  Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an diverse array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies (MVBs) that incorporate viral products, including dense bodies (DBs) and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in primary human microvascular endothelial cells (HMVECs) were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that UL32-GFP containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. However, UL32-GFP containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non- canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV, which may reflect distinct pathways for virus egress.Human cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life-long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with age-related pathology. HCMV infects many cells in the human host and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact how progeny virus is trafficked out of infected cells.
• Wu, M. H., Padilla-Rodriguez, M., Blum, I., Camenisch, A., Figliuolo da Paz, V., Ollerton, M., Muller, J., Momtaz, S., Mitchell, S. A., Kiela, P., Thorne, C., Wilson, J. M., & Cox, C. M. (2021). Proliferation in the developing intestine is regulated by the endosomal protein Endotubin. Developmental biology, 480, 50-61.
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  During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTB mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.
• Blum, I. R., Behling-Hess, C., Padilla-Rodriguez, M., Momtaz, S., Cox, C., & Wilson, J. M. (2020). Rab22a regulates the establishment of epithelial polarity. Small GTPases, 1-12.
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  Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.
• Jamwal, D. R., Laubitz, D., Harrison, C. A., Figliuolo da Paz, V., Cox, C. M., Wong, R., Midura-Kiela, M., Gurney, M. A., Besselsen, D. G., Setty, P., Lybarger, L., Bhattacharya, D., Wilson, J. M., Ghishan, F. K., & Kiela, P. R. (2020). Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice. Gastroenterology, 159(4), 1342-1356.e6.
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  Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis.
• Engevik, A. C., Faust, J. J., Goldenring, J. R., Kaji, I., Tyska, M. J., & Wilson, J. M. (2019). 117 – Syndapin 2 is Required for Microvillus Inclusion Formation Via Apical Bulk Endocytosis in Mice Lacking Myosin Vb. Gastroenterology, 156(6), S-31. doi:10.1016/s0016-5085(19)36851-9
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  from a GES.Frequency, amplitude and FAR appear to be reliable measures of EGG.In the setting of patients with Sx of gastroparesis undergoing temporary GES, cutaneous EGGs can provide safe and cost-effective means to measure electrophysiological properties and have significant correlation with important clinical measures.
• Engevik, A., Kaji, I., Faust, J., Meyer, A., Williams, J., Goldenring, J., Wilson, J. M., & Tyska, M. (2019). Loss of Myosin Vb promotes apical bulk endocytosis in neonatal enterocytes. Journal of Cell Biology, 218, 3667-3662. doi:10.1083/jcb.201902063
• Figliuolo da Paz, V., Jamwal, D. R., Gurney, M. T., Midura-Kiela, M. T., Harrison, C. A., Cox, C. M., Wilson, J. M., Ghishan, F. K., & Kiela, P. R. (2019). Rapid downregulation of DAB2 by toll-like receptor activation contributes to a pro-inflammatory switch in activated dendritic cells.. Frontiers in Immunology.
• Figliuolo da Paz, V., Jamwal, D. R., Gurney, M., Midura-Kiela, M., Harrison, C. A., Cox, C., Wilson, J. M., Ghishan, F. K., & Kiela, P. R. (2019). Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells. Frontiers in immunology, 10, 304.
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  Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11bCD103 DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4 cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of , and , and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.
• Cox, C. M., Lu, R., Salcin, K., & Wilson, J. M. (2017). The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation. Cellular and molecular gastroenterology and hepatology, 5(2), 145-156.
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  During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown.
• Cox, C. M., Lu, R., Salcin, K., & Wilson, J. M. (2018). The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation.. Cellular and molecular gastroenterology and hepatology, 5(2), 145-156. doi:10.1016/j.jcmgh.2017.11.001
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  During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown..By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy..Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking..Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.
• Cox, C. M., Parker, S. S., & Wilson, J. M. (2018). Rabs set the stage for polarity.. Small GTPases, 9(1-2), 116-129. doi:10.1080/21541248.2016.1277840
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  Cell polarity refers to the asymmetric localization of cellular components that allows cells to carry out their specialized functions, be they epithelial barrier function, transmission of action potentials in nerve cells, or modulation of the immune response. The establishment and maintenance of cell polarity requires the directed trafficking of membrane proteins and lipids - essential processes that are mediated by Rab GTPases. Interestingly, several of the Rabs that impact polarity are present in the earliest eukaryotes, and the Rab polarity repertoire has expanded as cells have become more complex. There is a substantial conservation of Rab function across diverse cell types. Rabs act through an assortment of effector proteins that include scaffolding proteins, cytoskeletal motors, and other small GTPases. In this review we highlight the similarities and differences in Rab function for the instruction of polarity in diverse cell types.
• Lybarger, L. P., Cox, C. M., Ghishan, F. K., Gurney, M. A., Harrison, C. A., Jamwal, D. R., Kiela, P. R., Laubitz, D., Midura-kiela, M. T., & Wilson, J. M. (2018). 820 - Intestinal Epithelial Mhcii Expression Modulates the Course of Autoimmune and Infectious Colitis in a Mouse Model of Conditional I-AB Knockout. Gastroenterology, 154(6), S-169-S-170. doi:10.1016/s0016-5085(18)30978-8
• Zeltzer, S., Zeltzer, C. A., Igarashi, S., Wilson, J., Donaldson, J. G., & Goodrum, F. (2018). Virus Control of Trafficking from Sorting Endosomes. mBio, 9(4).
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  The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates. Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV infection.
• Johnson, D. L., Wayt, J., Wilson, J. M., & Donaldson, J. G. (2017). Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking. Journal of cell science, 130(14), 2405-2415.
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  Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.
• Parker, S. P., Cox, C., & Wilson, J. M. (2017). Rabs set the stage for polarity. Small GTPases.
• Lu, R., & Wilson, J. M. (2016). Rab14 specifies the apical membrane through Arf6-mediated regulation of lipid domains and Cdc42. Scientific reports, 6, 38249.
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  The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase α [PtdIns(4)P5K] activator Arf6 or PtdIns(4)P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14's reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity.
• Bughio, F., Umashankar, M., Wilson, J., & Goodrum, F. (2015). Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells. Journal of virology, 89(13), 6536-50.
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  Endothelial cells (ECs) are a critical target of viruses, and infection of the endothelium represents a defining point in viral pathogenesis. Human cytomegalovirus (HCMV), the prototypical betaherpesvirus, encodes proteins specialized for entry into ECs and delivery of the genome to the nuclei of ECs. Virus strains competent to enter ECs replicate with differing efficiencies, suggesting that the virus encodes genes for postentry tropism in ECs. We previously reported a specific requirement for the UL133/8 locus of HCMV for replication in ECs. The UL133/8 locus harbors four genes: UL133, UL135, UL136, and UL138. In this study, we find that while UL133 and UL138 are dispensable for replication in ECs, both UL135 and UL136 are important. These genes are not required for virus entry or the expression of viral genes. The phenotypes associated with disruption of either gene reflect phenotypes observed for the UL133/8NULL virus, which lacks the entire UL133/8 locus, but are largely distinct from one another. Viruses lacking UL135 fail to properly envelop capsids in the cytoplasm, produce fewer dense bodies (DB) than the wild-type (WT) virus, and are unable to incorporate viral products into multivesicular bodies (MVB). Viruses lacking UL136 also fail to properly envelop virions and produce larger dense bodies than the WT virus. Our results indicate roles for the UL135 and UL136 proteins in commandeering host membrane-trafficking pathways for virus maturation. UL135 and UL136 represent the first HCMV genes crucial for early- to late-stage tropism in ECs.
• Cox, C. M., Mandell, E. K., Stewart, L., Lu, R., Johnson, D. L., McCarter, S. D., Tavares, A., Runyan, R., Ghosh, S., & Wilson, J. M. (2015). Endosomal regulation of contact inhibition through the AMOT:YAP pathway. Molecular biology of the cell, 26(14), 2673-84.
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  Contact-mediated inhibition of cell proliferation is an essential part of organ growth control; the transcription coactivator Yes-associated protein (YAP) plays a pivotal role in this process. In addition to phosphorylation-dependent regulation of YAP, the integral membrane protein angiomotin (AMOT) and AMOT family members control YAP through direct binding. Here we report that regulation of YAP activity occurs at the endosomal membrane through a dynamic interaction of AMOT with an endosomal integral membrane protein, endotubin (EDTB). EDTB interacts with both AMOT and occludin and preferentially associates with occludin in confluent cells but with AMOT family members in subconfluent cells. EDTB competes with YAP for binding to AMOT proteins in subconfluent cells. Overexpression of the cytoplasmic domain or full-length EDTB induces translocation of YAP to the nucleus, an overgrowth phenotype, and growth in soft agar. This increase in proliferation is dependent upon YAP activity and is complemented by overexpression of p130-AMOT. Furthermore, overexpression of EDTB inhibits the AMOT:YAP interaction. EDTB and AMOT have a greater association in subconfluent cells compared with confluent cells, and this association is regulated at the endosomal membrane. These data provide a link between the trafficking of tight junction proteins through endosomes and contact-inhibition-regulated cell growth.
• Lu, R., Dalgalan, D., Mandell, E. K., Parker, S. S., Ghosh, S., & Wilson, J. M. (2015). PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels. Molecular biology of the cell, 26(8), 1523-31.
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  PKCι is essential for the establishment of epithelial polarity and the normal assembly of tight junctions. We find that PKCι knockdown does not compromise the steady-state distribution of most tight junction proteins but results in increased transepithelial resistance (TER) and decreased paracellular permeability. Analysis of the levels of tight junction components demonstrates that claudin-2 protein levels are decreased. However, other tight junction proteins, such as claudin-1, ZO-1, and occludin, are unchanged. Incubation with an aPKC pseudosubstrate recapitulates the phenotype of PKCι knockdown, including increased TER and decreased levels of claudin-2. In addition, overexpression of PKCι results in increased claudin-2 levels. ELISA and coimmunoprecipitation show that the TGN/endosomal small GTPase Rab14 and PKCι interact directly. Immunolabeling shows that PKCι and Rab14 colocalize in both intracellular puncta and at the plasma membrane and that Rab14 expression is required for normal PKCι distribution in cysts in 3D culture. We showed previously that knockdown of Rab14 results in increased TER and decreased claudin-2. Our results suggest that Rab14 and aPKC interact to regulate trafficking of claudin-2 out of the lysosome-directed pathway.
• Wilson, J. M. (2015). The scaffolding protein GOPC regulates tight junction structure. Cell and Tissue Research.
• Lu, R., Johnson, D. L., Stewart, L., Waite, K., Elliott, D., & Wilson, J. M. (2014). Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis. Molecular biology of the cell, 25(11), 1744-54.
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  Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin-Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.
• Parker, S. S., Mandell, E. K., Hapak, S. M., Maskaykina, I. Y., Kusne, Y., Kim, J., Moy, J. K., St John, P. A., Wilson, J. M., Gothard, K. M., Price, T. J., & Ghosh, S. (2013). Competing molecular interactions of aPKC isoforms regulate neuronal polarity. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14450-5.
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  Atypical protein kinase C (aPKC) isoforms ζ and λ interact with polarity complex protein Par3 and are evolutionarily conserved regulators of cell polarity. Prkcz encodes aPKC-ζ and PKM-ζ, a truncated, neuron-specific alternative transcript, and Prkcl encodes aPKC-λ. Here we show that, in embryonic hippocampal neurons, two aPKC isoforms, aPKC-λ and PKM-ζ, are expressed. The localization of these isoforms is spatially distinct in a polarized neuron. aPKC-λ, as well as Par3, localizes at the presumptive axon, whereas PKM-ζ and Par3 are distributed at non-axon-forming neurites. PKM-ζ competes with aPKC-λ for binding to Par3 and disrupts the aPKC-λ-Par3 complex. Silencing of PKM-ζ or overexpression of aPKC-λ in hippocampal neurons alters neuronal polarity, resulting in neurons with supernumerary axons. In contrast, the overexpression of PKM-ζ prevents axon specification. Our studies suggest a molecular model wherein mutually antagonistic intermolecular competition between aPKC isoforms directs the establishment of neuronal polarity.
• Wilson, A. H., McCarter, S. D., Johnson, D. L., Kitt, K. N., Donohue, C., Adams, A., & Wilson, A. H. (2010). Regulation of tight junction assembly and epithelial polarity by a resident protein of apical endosomes. Traffic (Copenhagen, Denmark), 11(6).
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  The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C-terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three-dimensional culture. In addition, in cells overexpressing the C-terminal domain of endotubin, we observe a delay in re-establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.
• Wilson, A. H., Kitt, K. N., Hernández-Deviez, D., Ballantyne, S. D., Spiliotis, E. T., Casanova, J. E., & Wilson, A. H. (2008). Rab14 regulates apical targeting in polarized epithelial cells. Traffic (Copenhagen, Denmark), 9(7).
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  Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.
• Wilson, A. H., Hernández-Deviez, D., Mackay-Sim, A., & Wilson, A. H. (2007). A Role for ARF6 and ARNO in the regulation of endosomal dynamics in neurons. Traffic (Copenhagen, Denmark), 8(12).
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  During development, neuronal processes extend, branch and navigate to ultimately synapse with target tissue. We have shown a regulatory role for ARNO and ARF6 in dendritic branching and axonal elongation and branching during neuritogenesis, particularly with respect to cytoskeletal dynamics. Here, we have examined the role of ARF6 and the ARF GEF ARNO in endosomal dynamics during neurite elongation in hippocampal neurons. Axonal and dendritic endosomes were labeled by expression of the endosomal marker, endotubin. Expression of endotubin-green fluorescent protein resulted in targeting to tubular-vesicular structures throughout the somatodendritic and axonal domains. These endosomal structures did not colocalize with conventional early or late endosomal markers or with the synaptic vesicle marker, SV2. However, they did label with internalized lectin, indicating that they are endosomal structures. Expression of catalytically inactive ARNO (ARNO-E156K) or inactive ARF6 (ARF6-T27N) caused a redistribution of endotubin to the cell surface of the axons and dendrites. In contrast, expression of these constructs had no effect upon the distribution of SV2-positive structures. Furthermore, expression of inactive ARF1 (ARF1-T31N) did not change endotubin distribution. These results suggest that endotubin labels a distinct endosomal structure in neurons and that ARNO and ARF6 mediate neurite extension through the regulation of this compartment.
• Wilson, A. H., Hernández-Deviez, D. J., & Wilson, A. H. (2005). Functional assay of ARNO and ARF6 in neurite elongation and branching. Methods in enzymology, 404.
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  During development of the nervous system, neurite outgrowth is necessary for the formation of connections between nerve cells. Neurons are highly polarized cells that send out distinct processes, axons, and dendrites; however, the molecular regulation of the differential growth of these processes remains incompletely understood. Primary cultures of rat hippocampal neurons have been used to study many aspects of neuronal cell biology, including neurite extension, establishment of polarity, biogenesis of synapses, and membrane trafficking. After attachment to the substrate, hippocampal neurons begin sending out multiple processes by approximately 12 h after plating. The axonal process is derived from one of these processes, and is evident after 48 h in culture. Complete polarity of axons and dendrites is established after 7 days in culture. The establishment of these cultures and the ability to transfect them with potential regulatory genes allows the researcher to dissect out the pathways relevant to neurite extension. To study the role of small GTPases in neurite extension and branching, we describe methods for culture of hippocampal neurons, for transfection of these cells, and assessment of neurite extension and branching.
• Wilson, A. H., Hernández-Deviez, D. J., Roth, M. G., Casanova, J. E., & Wilson, A. H. (2004). ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha. Molecular biology of the cell, 15(1).
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  In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking. To investigate the role of these molecules in axonogenesis, we expressed ARNO and ARF6 in cultured rat hippocampal neurons. Expression of catalytically inactive ARNO or dominant negative ARF6 resulted in enhanced axonal extension and branching and this effect was abrogated by coexpression of constitutively active ARF6. We sought to identify the downstream effectors of ARF6 during neurite extension by coexpressing phosphatidyl-inositol-4-phosphate 5-Kinase alpha [PI(4)P 5-Kinase alpha] with catalytically inactive ARNO and dominant negative ARF6. We found that PI(4)P 5-Kinase alpha plays a role in neurite extension and branching downstream of ARF6. Also, expression of inactive ARNO/ARF6 depleted the actin binding protein mammalian ena (Mena) from the growth cone leading edge, indicating that these effects on axonogenesis may be mediated by changes in cytoskeletal dynamics. These results suggest that ARNO and ARF6, through PI(4)P 5-Kinase alpha, regulate axonal elongation and branching during neuronal development.
• Wilson, A. H., Hernández-Deviez, D. J., Casanova, J. E., & Wilson, A. H. (2002). Regulation of dendritic development by the ARF exchange factor ARNO. Nature neuroscience, 5(7).
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  Here we analyzed the role of ARF6, a member of the ADP-ribosylation factor (ARF) family of small GTPases, in dendritic arbor development in rat hippocampal neurons in culture. Overexpression of the inactive form of the GTP exchange factor ARNO (ARF nucleotide binding site opener) or inactive ARF6 enhanced dendritic branching, whereas coexpression of either Rac1 (a member of the Rho family of small GTPases known to control dendritic dynamics and growth) or active ARF6 with inactive ARNO eliminated the enhanced branching effect. These results indicate that the ARF family of small GTPases contributes to the regulation of dendritic branching, and that ARF6 activation turns on two independent pathways that suppress dendritic branching in vivo: one through Rac1 and the other through ARF6.
• Brown, J. K., Gottlieb, Y., Hunter, M. S., Karr, T. L., Kelly, S. E., Wilson, J. M., & Zchori-Fein, E. (2001). A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps. Proceedings of the National Academy of Sciences, 98(22), 12555-12560. doi:10.1073/pnas.221467498
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  The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia, a genus of parasitoid wasps. Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the "Encarsia bacterium" (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia. Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia. Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.
• Wilson, J., Gokay, K. E., Young, R. S., & Wilson, J. M. (2001). Cytoplasmic signals mediate apical early endosomal targeting of endotubin in MDCK cells. Traffic (Copenhagen, Denmark), 2(7).
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  Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin-Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.
• Dotti, C. G., Hoop, M. D., Parton, R. G., Toh, B. H., Wilson, J. M., & Zorzi, N. (2000). EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts.. Molecular biology of the cell, 11(8), 2657-71. doi:10.1091/mbc.11.8.2657
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  EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.
• Gokay, K. E., & Wilson, J. M. (2000). Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs. Traffic, 1(Issue 4). doi:10.1034/j.1600-0854.2000.010408.x
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  The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin-Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule. Copyright © Munksgaard 2000.
• Wilson, J., Garver, W. S., Heidenreich, R. A., Erickson, R. P., Thomas, M. A., & Wilson, J. M. (2000). Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments. Journal of lipid research, 41(5).
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  Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.
• Wilson, J., Gokay, K. E., & Wilson, J. M. (2000). Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs. Traffic (Copenhagen, Denmark), 1(4).
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  The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin-Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.
• Falk, T., Garver, W., Erickson, R., Wilson, J., & Yool, A. (1999). Expression of Niemann-Pick type C transcript in rodent cerebellum in vivo and in vitro. Brain Research, 839(1). doi:10.1016/S0006-8993(99)01678-9
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  This study assesses the developmental expression of the Niemann-Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human (NPC1) and mouse (Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.
• Yool, A. J., Erickson, R. P., Falk, T., Wilson, J. M., Garver, W. S., Erickson, R. P., Garver, W. S., Wilson, J. M., Yool, A. J., & Falk, T. (1999). Expression of Niemann-Pick type C transcript in rodent cerebellum in vivo and in vitro.. Brain research, 839(1), 49-57. doi:10.1016/s0006-8993(99)01678-9
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  This study assesses the developmental expression of the Niemann-Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human (NPC1) and mouse (Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.
• Wilson, J., Allen, K., Gokay, K. E., Thomas, M. A., Speelman, B. A., & Wilson, J. M. (1998). Biosynthesis of endotubin: an apical early endosomal glycoprotein from developing rat intestinal epithelial cells. The Biochemical journal, 330 ( Pt 1).
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  Endosomes are the site of sorting of internalized receptors and ligands in all cell types and, in polarized cells, the apical endosomal compartment is involved in the selective transepithelial transport of immunoglobulins and growth factors. The biochemical composition of this specialized compartment remains largely unresolved. We have characterized a glycoprotein, called endotubin, that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. A monoclonal antibody against endotubin recognizes a broad band of 55-60kDa, which upon isoelectric focusing can be resolved into two bands, and a faint band of 140kDa. Metabolic labelling followed by immunoprecipitation indicates that endotubin is synthesized as a 140kDa precursor that is cleaved to the 55-60kDa forms. High pH washing of endosomal membranes removes the 55-60kDa forms from the membrane, whereas the high-molecular-mass form remains membrane associated and appears to be an integral membrane protein. Immunoblotting with a polyclonal antibody against the putative cytoplasmic tail of the protein identifies a 140kDa band and a band of 74kDa, presumably the cleavage product. Immunoprecipitation with antibodies against the 55-60kDa form results in coprecipitation of a 74kDa protein, and immunoprecipitation with antibody against the 74kDa protein results in coprecipitation of the 55-60kDa form. Epitope mapping of the monoclonal antibody binding site supports a proposed type I membrane protein orientation. We propose that endotubin is proteolytically processed into a heterodimer with the 55-60kDa fragment remaining membrane-associated through a non-covalent association with the membrane-bound 74kDa portion of the molecule.
• Colton, T. L., & Wilson, J. M. (1997). Targeting of an intestinal apical endosomal protein to endosomes in nonpolarized cells.. The Journal of cell biology, 136(2), 319-30. doi:10.1083/jcb.136.2.319
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  Polarized cells such as epithelial cells and neurons have distinct endosomal compartments associated with different plasma membrane domains. The endosomes of the neuronal cell body and the basolateral cytoplasm of epithelial cells are thought to perform cellular "housekeeping" functions such as the uptake of nutrients and metabolites, while the endosomes in the apical cytoplasm or axons are thought to be specialized for the sorting and transcytosis of cell type-specific ligands and receptors. However, it is not known if nonpolarized cells such as fibroblasts contain a specialized endosomal compartment analogous to the specialized endosomes found in neurons and epithelia. We have expressed a protein that is normally found in the apical early endosomes of developing intestinal epithelial cells in normal rat kidney fibroblasts. This apical endosomal marker, called endotubin, is targeted to early endosomes in transfected fibroblasts, and is present in peripheral as well as perinuclear endosomes. The peripheral endosomes that contain endotubin appear to exclude transferrin, fluid phase markers, and the mannose-6-phosphate receptor, although in the perinuclear region colocalization of endotubin and these markers is present. In addition, endotubin positive structures do not tubulate in response to brefeldin A and instead redistribute to a diffuse perinuclear location. Since this endosomal compartment has many of the characteristics of an apical or axonal endosomal compartment, our results indicate that nonpolarized cells also contain a specialized early endosomal compartment.
• Erickson, R. P., Colton, T. L., Garver, W. S., Heidenreich, R. A., Hossain, G. S., Kozloski, M. A., Kozloski, K., & Wilson, J. M. (1997). Altered expression of caveolin-1 and increased cholesterol in detergent insoluble membrane fractions from liver in mice with Niemann-Pick disease type C.. Biochimica et biophysica acta, 1361(3), 272-80. doi:10.1016/s0925-4439(97)00047-1
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  Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.
• Garver, W., Erickson, R., Wilson, J., Colton, T., Hossain, G., Kozloski, M., & Heidenreich, R. (1997). Altered expression of caveolin-1 and increased cholesterol in detergent insoluble membrane fractions from liver in mice with Niemann-Pick disease type C. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1361(3). doi:10.1016/S0925-4439(97)00047-1
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  Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.
• Neutra, M. R., Trahair, J. F., & Wilson, J. M. (1995). Identification of a marker antigen for the endocytic stage of intestinal development in rat, sheep, and human.. Journal of pediatric gastroenterology and nutrition, 21(3), 277-87. doi:10.1097/00005176-199510000-00005
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  Vacuolated enterocytes are highly endocytic epithelial cells present in intestines of diverse mammalian species during neonatal and/or fetal development. Using monoclonal antibodies raised against membrane fractions, we previously identified a 55-61 kd membrane glycoprotein that is restricted to apical endosomal tubules of vacuolated enterocytes in fetal and suckling rats. To determine whether this cell-specific antigen is present in vacuolated enterocytes of fetal sheep or humans, the endosomal antigen was immuno-affinity purifed from rats and used to generate and purify specific rabbit polyclonal antibodies. Light microcopic and electron microscopic immunocytochemistry showed that antigens cross-reactive with the rat endosomal antigen are present in vacuolated enterocytes of fetal sheep and fetal human small intestine and are restricted to apical endosomal tubules in these cells. Immunoblot analysis of tissue extracts from fetal human intestines showed antigen(s) at 55 and 60 kd, as well as a major form at 130 kd. Cross-reactive antigen(s) from fetal sheep intestines appeared as 42- and 50-kd bands. Although the molecular identities of the sheep and human antigens are not yet established, these results show that these antigens can serve as markers for the endocytic state of intestinal developmemt in humans as well as other mammals.
• Wilson, J., Speelman, B. A., Allen, K., Grounds, T. L., Neutra, M. R., Kirchhausen, T., & Wilson, J. M. (1995). Molecular characterization of an apical early endosomal glycoprotein from developing rat intestinal epithelial cells. The Journal of biological chemistry, 270(4).
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  The apical endosomal compartment is thought to be involved in the sorting and selective transport of receptors and ligands across polarized epithelia. To learn about the protein components of this compartment, we have isolated and sequenced a cDNA that encodes a glycoprotein that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. The deduced amino acid sequence predicts a protein of 1216 amino acids with a molecular mass of 133,769 Da. The deduced amino acid sequence together with amino-terminal amino acid sequencing indicate that there is a cleaved 21-amino acid signal sequence at the NH2-terminal portion of the molecule. There is a single hydrophobic region near the carboxyl terminus that has the characteristics of a membrane-spanning domain and a 36-amino acid cytoplasmic tail. We have found that the major form of this protein in intestinal epithelial cells has a molecular mass of 55-60 kDa, which is significantly smaller than the size predicted from the cDNA sequence, suggesting that the protein is synthesized as a large precursor and processed to the smaller form. The smaller form remains associated with the membrane, however, possibly through noncovalent association with the transmembrane portion of the molecule or with another membrane protein. The extracytoplasmic domain is cysteine-rich, with three cysteine-rich repeats that are similar to cysteine repeats present in several receptor proteins. However, there is no other significant similarity to other proteins in the GenBank. The cytoplasmic tail contains a possible internalization motif and several consensus motifs for serine/threonine kinases. Northern blot analysis suggests a single abundant message, and Southern blot analysis is consistent with a single gene and the absence of pseudogenes for this unique endosomal protein.
• Dvorak, B., Holubec, H., Koldovsky, O., Lebouton, A. V., & Wilson, J. M. (1994). Epidermal growth factor and transforming growth factor-alpha mRNA in rat small intestine: in situ hybridization study.. FEBS letters, 352(3), 291-5. doi:10.1016/0014-5793(94)00942-2
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  The expression of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) mRNA in the small intestine of suckling and adult rats was examined by in situ hybridization. EGF mRNA was found mainly in the intestinal crypts in adult rats. Adult rats also exhibited a considerably stronger signal for EGF mRNA in comparison to suckling rats, where the signal was very low or absent. In contrast to EGF, very strong expression of TGF-alpha mRNA was observed in the small intestine of both adult and suckling rats. These data suggest the differences between the expression of EGF and TGF-alpha in the developing small intestine.
• Dvorak, B., Koldovsky, O., Lebouton, A. V., & Wilson, J. M. (1994). 70 LOCALIZATION OF EGF AND TGF-α mRNA IN THE SMALL INTESTINE OF SUCKLING AND ADULT RATS. Journal of Pediatric Gastroenterology and Nutrition, 19(3), 346. doi:10.1097/00005176-199410000-00082
• Neutra, M. R., Whitney, J. A., & Wilson, J. M. (1991). Biogenesis of the apical endosome-lysosome complex during differentiation of absorptive epithelial cells in rat ileum.. Journal of Cell Science, 100(1), 133-143. doi:10.1242/jcs.100.1.133
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  Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20-22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosome-lysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments.
• Neutra, M. R., Whitney, J. A., & Wilson, J. M. (1991). Biogenesis of the apical endosome-lysosome complex during differentiation of absorptive epithelial cells in rat ileum.. Journal of cell science, 100 ( Pt 1)(1), 133-43.
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  Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20-22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosome-lysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments.
• Wilson, J., Whitney, J., & Neutra, M. (1991). Biogenesis of the apical endosome-lysosome complex during differentiation of absorptive epithelial cells in rat ileum. Journal of Cell Science, 100(1).
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  Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20-22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosomelysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments.
• Fasel, N., Kraehenbuhl, J. P., & Wilson, J. M. (1990). Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.. Journal of cell science, 96 ( Pt 1)(1), 143-9.
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  Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.
• Fasel, N., Kraehenbuhl, J., & Wilson, J. M. (1990). Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.. Journal of Cell Science, 96(1), 143-149. doi:10.1242/jcs.96.1.143
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  Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.
• Wilson, J., Fasel, N., & Kraehenbuhl, J. (1990). Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells. Journal of Cell Science, 96(1).
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  Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 μm frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool.Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail) In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.
• Amerongen, H. M., Mack, J. A., Neutra, M. R., & Wilson, J. M. (1989). Membrane domains of intestinal epithelial cells: distribution of Na+,K+-ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation.. The Journal of cell biology, 109(5), 2129-38. doi:10.1083/jcb.109.5.2129
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  The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.
• Kraehenbuhl, J. P., Neutra, M. R., Weltzin, R. A., & Wilson, J. M. (1988). Membrane domains and macromolecular transport in intestinal epithelial cells.. The American review of respiratory disease, 138(6 Pt 2), S10-6. doi:10.1164/ajrccm/138.6_pt_2.s10
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  Epithelial cell plasma membranes are organized as biochemically and functionally distinct domains and subdomains. We have explored the composition and maintenance of specific membrane regions during endocytosis and transepithelial vesicular transport in specialized cells of the intestinal epithelium. A unique membrane glycoprotein has been identified in apical endocytic subdomains and endosomal tubules of absorptive cells in suckling rat ileum. This endocytic system is involved in sorting and transepithelial transport of peptide growth factors as well as delivery of milk macromolecules to lysosomes. In M cells of follicle-associated epithelium, we have shown that membrane-bound proteins are efficiently transported to a specialized subdomain of basolateral membrane. The apical membranes of these cells bear immunoglobulin binding sites and transport monoclonal IgA antibodies to the basolateral side, where they may interact with cells of the mucosal immune system.
• Neutra, M. R., Whitney, J. A., & Wilson, J. M. (1987). Identification of an endosomal antigen specific to absorptive cells of suckling rat ileum.. The Journal of cell biology, 105(2), 691-703. doi:10.1083/jcb.105.2.691
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  A membrane fraction enriched in apical endosomal tubules was isolated from absorptive cells of suckling rat ileum and used as an immunogen to generate anti-endosome monoclonal antibodies. By immunofluorescence, one of these antibodies bound exclusively to the region of the apical endocytic complex in ileal absorptive cells, but not to other cell types. Immunoblot analysis showed the antigen as a diffuse 55-61-kD band which was highly enriched in the endosome fraction over whole-cell homogenate. The antigen appears to be an intramembrane glycoprotein: it partitioned primarily in the detergent phase after TX-114 extraction, and shifted to 44 kD after chemical deglycosylation. EM immunocytochemistry showed that the antibody bound to the luminal side of endosomal tubule membranes, a portion of endosomal vesicle membranes, and in endocytic pits of apical plasma membranes. However, it did not bind to multivesicular bodies, the giant lysosome, or other organelles. Immunocytochemistry after uptake with adsorbed or soluble tracer proteins showed that the antigen labeled portions of both prelysosomal pathways previously described in these cells (Gonnella, P.A., and M. R. Neutra, 1984, J. Cell Biol., 99:909-917). The function of this glycoprotein is not known, but inasmuch as it has been detected only in absorptive cells of suckling rat ileum, it may serve a function specific to these cells. Nevertheless, this endosomal antigen, designated glycoprotein (gp) 55-61, will serve as a useful marker for exploring membrane dynamics in early stages of the endocytic pathway.
• Wilson, J., & King, B. (1986). Sorting and transepithelial transport of adsorbed protein tracers: Effects of temperature. The Anatomical Record, 216(1). doi:10.1002/ar.1092160106
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  The epithelium of the guinea pig yolk sac is involved in the selective transport of macromolecules to the fetus. We studied the compartments involved in sorting and transepithelial transport of protein tracers and the effect of lowered temperature (18°C) on these events. Explants of yolk sac were incubated with a mixture of cationized ferritin (CF) and horseradish peroxidase (HRP, Sigma type VI). At 4°C, both tracers were bound to the cell surface and binding of an HRP‐gold complex was shown to be inhibited by mannan. At 37°C and 18°C, both tracers were taken up into tubules and vesicles in the apical cytoplasm. Usually the tubules contained a mixure of tracers, but they often showed a polarized distribution with CF and HRP at opposite ends. The vesicles also contained mixtures of the tracers, but some contained only one. In addition, there were some irregularly shaped vacuoles composed of saccules that contained either a mixture, HRP alone, or CF alone. These results suggest that these adsorbed ligands are binding to unique microdomains of the endocytic complex. After 20 min at 37°C coated vesicles 100 nm in diameter were located in the apical cytoplasm and coated vesicles of the same size were located at the lateral cell membrane. Usually they contained only HRP or CF, although occasional mixtures were seen. At 18°C, HRP was transported across the cells in 100 nm vesicles. However, transport of CF was completely inhibited at the lower temperature. Although uptake at 18°C was slower than that at 37°C, experiments with 125I‐CF allowed comparison of time points at which equal uptake had occurred. These experiments demonstrated that inhibition of CF transport was not due to decreased amounts of internalized CF. These differences in transport of HRP and CF suggest that multiple compartments are involved in the sorting and transepithelial transport of protein, and at least one is perturbed by lower temperature. Copyright © 1986 Wiley‐Liss, Inc.
• Wilson, J., & King, B. (1985). Transport of horseradish peroxidase across monkey trophoblastic epithelium in coated and uncoated vesicles. The Anatomical Record, 211(2). doi:10.1002/ar.1092110209
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  This study used membranous chorion of the macaque monkey placenta to examine uptake and processing of exogenous proteins. Tissue was incubated with either cationic or anionic horseradish peroxidase. Incubation time was varied between 5–25 min to follow the endocytic pathways. In spite of some differences in binding, uptake and processing of the isozymes was similar. In the presence of tracers at 37°C both horseradish peroxidases were taken up in large (150–175) nm diameter coated vesicles. In addition, coated tubules 300–400 nm in length and 50–100 nm in diameter were seen in the apical cytoplasm. Often, the tubules bud off small (85–105 nm diameter) protein‐filled coated vesicles which traversed the cytoplasm and fused with the basal‐lateral plasma membrane. In other cases, the tubules or vesicles lost their clathrin coats and fused to form larger endocytic vesicles which later fused with phagolysosomes. After longer incubation, larger uncoated vesicles (endosomes) were seen releasing their contents at the basal‐lateral membrane. These results suggest that multiple transport pathways exist in this epithelium. The first, involving only coated structures, may function to sort and concentrate specific ligands important for embryonic development. The second, involving the formation and translocation of large uncoated vesicles to the basal‐lateral membrane, may also provide nutrients to the embryo. A third pathway directs the protein to phagolysosomes where it is presumably degraded. Degradation products could be used by the cells of the membranous chorion or may provide nutrients to the embryo. Copyright © 1985 Wiley‐Liss, Inc.
• King, B., & Wilson, J. (1983). A fine structural and cytochemical study of the rhesus monkey yolk sac: Endoderm and mesothelium. The Anatomical Record, 205(2). doi:10.1002/ar.1092050205
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  This study has examined the fine structure and some cytochemical characteristics of the endodermal and mesothelial cells of the rhesus monkey yolk sac between 25 and 66 days of gestation. The endodermal cells were characterized by abundant granular endoplasmic reticulaum, some agranular endoplasmic reticulum, a well‐developed Golgi apparatus, and numerous large mitochondria. During the earlier part of the period studied, endodermal cells had a few acid phosphatase and arylsulfatase‐positive lysosomes and moderate numbers of catalase‐positive microperoxisomes. During the later stages of development, large granules (believed to be lysosomes) with a heterogeneous content were numerous in the cytoplasm. Mesothelial cells showed fewer development changes. Throughout this period they were usually flattened cells with long microvilli, small mitochondria, and limited amounts of granular endoplasmic reticulum. The mesothelial cells had acid phosphatase reaction product in the Golgi region and occasional large vesicles, but were negative for arylsulfatase and catalase. One specimen was incubated at 37°C in the presence of horseradish peroxidase in order to examine endocytosis. Both the mesothelial cells and endodermal cells internalized the peroxidase into a variety of cytoplasmic vesicles. Based on their cytology, the endodermal cells may function in the synthesis of serum proteins during this period, as has been suggested in other species. They may also be involved in lipid metabolism. The mesothelial cells appeared less synthetically active, but evidence suggested that they may be involved in collagen and extracellular matrix production. The endocytic activity displayed by both cell types may indicate a role in fluid and metabolite transfer across the epithelia. The cytology of both cell types was very similar to that described for human yolk sacs, suggesting that the rhesus monkey may be a useful species in which to study the maturation of yolk sac function. Copyright © 1983 Wiley‐Liss, Inc.